International Journal of Sport Nutrition and Exercise Metabolism, 2007, 17, 456-467

© 2007 Human Kinetics, Inc.

The Effects of Nutritional Supplementation

Throughout an Endurance Run on Leucine
Kinetics During Recovery
Sharon L. Miller, P. Courtney Gaine, Carl M. Maresh,
Lawrence E. Armstrong, Cara B. Ebbeling, Linda S. Lamont,
and Nancy R. Rodriguez

This study determined the effect of nutritional supplementation throughout endur-

ance exercise on whole-body leucine kinetics (leucine rate of appearance [Ra],
oxidation [Ox], and nonoxidative leucine disposal [NOLD]) during recovery. Five
trained men underwent a 2-h run at 65% VO2max, during which a carbohydrate
(CHO), mixed protein-carbohydrate (milk), or placebo (PLA) drink was consumed.
Leucine kinetics were assessed during recovery using a primed, continuous infusion
of 1-13C leucine. Leucine Ra and NOLD were lower for milk than for PLA. Ox
was higher after milk-supplemented exercise than after CHO or PLA. Although
consuming milk during the run affected whole-body leucine kinetics, the benefits
of such a practice for athletes remain unclear. Additional studies are needed to
determine whether protein supplementation during exercise can optimize protein
utilization during recovery.

Key Words: whole-body protein turnover, milk supplementation

Endurance exercise alters rates of whole-body leucine kinetics during exercise

and in the postexercise recovery period. During moderate- to high-intensity exercise
there is a decrease in protein synthesis (3, 26, 39) and increases in protein breakdown
(7, 24, 39) and leucine oxidation (3, 19, 24, 32). Because most endurance athletes
are able to maintain lean body mass and improve strength and performance, the
catabolic environment during exercise must be balanced by an anabolic phase during
the recovery period (6, 10). Studies of protein utilization after exercise have shown
increases in whole-body protein synthesis (3, 10, 26), whereas rates of breakdown
decrease or do not differ from those observed at rest (3, 10, 26). Losses in lean
body mass and strength can occur during heavy training periods if the proteolysis
occurring during exercise is not countered by a comparable increase in protein
synthesis postexercise. Nutritional interventions might be a way to optimize protein
utilization in athletes participating in vigorous endurance training (5, 16).

Miller, Gaine, and Ebbeling were and Rodriguez is with the Dept. of Nutritional Sciences, and Maresh
and Armstrong, the Dept. of Kinesiology, University of Connecticut, Storrs, CT 06269. Lamont is with
the Dept. of Exercise Science, University of Rhode Island, Kingston, RI 02881-0810.

456
 Leucine Kinetics During Recovery From Exercise   457

Carbohydrate supplementation during exercise is the classic example of a

nutrient intervention aimed at improving performance in endurance athletes.
Because of its ability to increase insulin levels (12, 30), maintain blood glucose
levels (12, 36), and spare muscle glycogen stores (40), carbohydrate supple-
mentation during exercise likely affects whole-body protein utilization, as well.
Providing carbohydrate during exercise can reduce the reliance on endogenous
protein stores for energy, thus attenuating leucine oxidation (3, 9, 14) and ammonia
production (30). Similarly, there is a relationship between glycogen stores and
protein utilization, with glycogen-depleted individuals exhibiting greater protein
breakdown (15) and branched-chain keto-acid dehydrogenase activity (36) than
carbohydrate-loaded or -supplemented subjects. Although some studies have
documented the relationship between carbohydrate intake and protein utiliza-
tion during endurance exercise, the effect of protein ingestion during exercise
on protein utilization has not been extensively studied.
Providing protein or amino acids influences protein utilization at rest and
the protein-related metabolic response to resistance exercise. At rest, amino acid
administration improves protein utilization (1, 22). Paddon-Jones et al. (23) observed
~60% increases in muscle protein-synthetic rates in young and old subjects after
they ingested 15 g of essential amino acids at rest. Protein administration (alone
or with carbohydrate) before, during, or after resistance exercise enhances protein
utilization in response to exercise (1, 2, 25, 33, 34). Biolo et al. (1) compared the
effects of amino acid infusion at rest and after resistance exercise, and, despite
similar increases in arterial amino acid concentrations, there were increased rates
of amino acid transport and a 2-fold increase in muscle protein synthesis after
exercise. Furthermore, the observed effects of amino acids in increasing muscle
protein synthesis postexercise have been noted with as little as 6 g of essential
amino acids (2, 25). Although it seems clear that providing amino acids at rest
and in association with resistance exercise improves protein utilization, there are
limited data regarding the interaction of protein intake and endurance exercise on
whole-body protein utilization.
Theoretically, ingesting amino acids during endurance exercise should attenuate
endogenous protein breakdown occurring in response to demands for gluconeogenic
precursors or metabolic intermediates during prolonged exercise (12, 20). In addi-
tion, an increase in plasma amino acids during exercise could set the stage for a
more anabolic recovery period, aiding in tissue repair after exercise (5, 29). A few
studies have investigated the metabolic impact of consuming a protein-carbohydrate
supplement during the recovery period and observed increases in whole-body and
leg protein gains (17), improved nitrogen balance (28), and enhanced glycogen
replenishment (41) after supplementation. The effects of protein ingestion during
endurance exercise on protein utilization during the recovery phase, however, have
not been delineated.
Koopman et al. (14), to the best of our knowledge, conducted the only study
that examined the effect of protein ingestion during endurance exercise on protein
utilization. They found that consumption of a protein-carbohydrate mixture during
6 h of endurance exercise at 50% VO2max in endurance athletes improved net protein
balance at rest, during exercise, and in the recovery period (14). Although protein
oxidation increased almost 2-fold, the ~60% decrease in protein breakdown con-
tributed to noted improvements in protein utilization in response to the supplement.
458   Miller et al.

Koopman et al. (14) used a protein-carbohydrate supplement that contained ~50 g

of carbohydrate and 18 g of protein per 30 min of exercise. The high energy and
solute content of this beverage would likely limit its routine use during endurance
exercise. The question remains, however, whether similar improvements in protein
utilization can be achieved with consumption of lesser amounts of carbohydrate
and protein during an endurance bout.
The current study was designed to compare the effects of consuming a carbohy-
drate and a mixed protein-carbohydrate beverage throughout an endurance-exercise
bout on whole-body leucine kinetics during the recovery period after exercise. We
hypothesized that consuming the protein-carbohydrate mix would improve pro-
tein utilization during the recovery period compared with consumption of either
carbohydrate alone or placebo.

Materials and Methods

Subjects
After project approval by the institutional review board at the University of Con-
necticut, 5 endurance-trained men (age 18–30 y) were recruited from the com-
munity to participate in the study. Each participant provided a complete medical
history, a training schedule, and a record of dietary intake. Subjects were required
to be running a minimum of 56 km/wk for inclusion in the study. Individuals
reporting metabolic or cardiovascular abnormalities, gastrointestinal disorders
(i.e., lactose intolerance), or use of nutritional or sports supplements or anabolic
steroids were excluded from the study. All subjects provided informed, written
consent. Subject characteristics are provided in Table 1.

Experimental Design
This study employed a randomized crossover design, with volunteers serving as
their own controls. After initial baseline testing, subjects participated in 3 exer-
cise trials during which a supplement containing carbohydrate (CHO), mixed
carbohydrate-protein (milk), or a nonnutritive placebo (PLA) was administered
during a 2-h endurance bout. The trials were separated by 7-d “wash-out” peri-
ods for isotope-decay recovery from one trial to the next. Vigorous exercise, as
well as consumption of alcohol and caffeine, was restricted on the day preceding
each trial.

Table 1  Subject Characteristics (N = 5)

Characteristic Value
Age (y) 22 ± 1.0
Height (cm) 178 ± 3.0
Weight (kg) 71 ± 3.0
Fat-free mass (kg) 62 ± 2.0
VO2max (mL.kg–1.min–1) 60 ± 0.9
Values are means ± standard error of the mean.
 Leucine Kinetics During Recovery From Exercise   459

Baseline Measures
Before initiation of the trials, subjects reported to the laboratory for preliminary data
collection. They underwent a test of maximal oxygen consumption (VO2max) and
then returned at a later time to determine the treadmill speed corresponding to 65%
of their predetermined VO2max. Body composition was determined by hydrostatic
weighing and calculated using the equations of Brozek et al. (4).

Dietary Records
Three-day food-intake records were obtained from subjects at baseline and for the
days leading up to each trial. Mean energy intake and macronutrient composition
of the diets were analyzed by a registered dietitian using Nutritionist IV software
(N2 Computing, Salem, OR).

Isotopes
Stocks of 1-13C-leucine (99% isotopic enrichment, Cambridge Isotope Laboratories,
Cambridge MA) were determined to be pyrogen free by a commercial laboratory
(South Mountain Labs, Lackawanna, NJ), and infusate was prepared aseptically
on the morning of each study.

Composition of Supplements
The CHO drink consisted of 45 g of dextrose (table sugar) mixed with 835 mL of
bottled water and nonsweetened flavoring (Kool-Aid, Kraft, Inc., White Plains, NY).
The milk supplement consisted of 480 mL of skim milk (17 g of protein and 27 g
of carbohydrate) diluted with 355 mL of bottled water. Flavoring and aspartame
(Equal, NutraSweet Co., Deerfield, IL) were added to the milk drink in an effort
to mask the nature of its composition and to increase palatability. The CHO and
protein drinks were isocaloric, providing ~170 kcal in total. The PLA drink was
prepared using bottled water, nonsweetened flavoring, and aspartame.

Study Protocol
Figure 1 illustrates the study protocol. Subjects arrived at the human-performance
laboratory at the University of Connecticut at ~7 AM after an overnight fast. Before
exercise a 19 G Teflon catheter (Jelco, Critikon, Tampa, FL) was inserted into a
superficial forearm vein for baseline blood sampling. An intravenous saline drip was
maintained to keep the line patent. Subjects then began running for 120 min at a speed
predetermined to elicit an effort equal to 65% of their individual VO2max. Water was
available for subjects to drink ad libitum throughout the endurance trial, and a fan was
positioned near the treadmill to maintain a comfortable environment. At 20, 40, 60,
and 80 min, subjects drank 200 mL of supplement while straddling the treadmill.

Assessment of Leucine Kinetics

After the exercise had been completed an additional catheter was placed in a con-
tralateral hand vein for arterialized blood sampling during the isotope infusion. An
intravenous saline drip was maintained to keep the line patent. The participant’s
460   Miller et al.

Figure 1 — Study protocol.

hand was placed in a Plexiglas box and was heated to ~70 °C so that arterialized
blood samples could be obtained (8). After collection of blood and breath samples
for background 1-13C-leucine enrichment, a primed continuous infusion of 1-13C-
leucine (4 µmol/kg, 4.8 µmol∙kg–1∙h–1) was initiated (Razel Syringe Pump, Razel
Scientific Instruments Inc., Stamford, CT) and continued for 180 min. At 120 min,
blood and breath measurements were collected at 15-min intervals for 1 h. Plasma
samples were spun in a refrigerated centrifuge (4 °C) at 2000 rpm for 10 min and
stored at –80 °C for subsequent analyses. For 13CO2 breath enrichments, subjects
breathed into a Douglas bag for 2 min at specified time intervals. Breath samples
were then transferred to 20-mL Venoject containers for isotope-ratio analysis.
Plasma 13C-KIC and 13CO2 enrichments were determined by gas chromatography
and isotope-ratio mass spectroscopy, respectively, by a commercial laboratory
(Metabolic Solutions, Nashua, NH). Blood 13C-KIC and breath 13CO2 data were
reviewed at 5 time points (120, 135, 150, 165, and 180 min) to confirm steady-state
conditions. Steady-state conditions were assumed when the coefficient of variation
of the atom-percentage-excess values at isotopic plateau was <10%. To account for
possible contributions of naturally occurring 13C in milk to expired 13CO2 enrich-
ment, 3 subjects were studied without concurrent infusion of 1-13C-leucine, and their
breath 13CO2 enrichments were determined at baseline (before milk consumption)
and at the same time periods after exercise in the current study. No differences were
noted in expired 13CO2 enrichments as a result of milk consumption.

Calculation of Leucine Kinetics

Data from the 5 time points were averaged for each subject, and group means
were determined. Leucine rate of appearance (leucine Ra; indicator of protein
breakdown), leucine oxidation (Ox), and nonoxidative leucine disposal (NOLD;
indicator of protein synthesis) were then calculated using the reciprocal-pool
model (11).
 Leucine Kinetics During Recovery From Exercise   461

Statistical Analysis
Primary outcome measures were leucine Ra, Ox, and NOLD in response to the 3
supplements. Treatment means were calculated for all variables and subjected to
a 3-way analysis of variance (ANOVA) with differences detected using Tukey’s
post hoc analysis with an α level set at 0.05. Data are expressed as mean ± stan-
dard error of the mean. Statistical analyses were conducted using SAS software
(Cary, NC).

Results
Dietary Intake
Energy intakes for the 3-d period preceding each trial and the single day before each
study did not differ for or between subjects. Likewise, macronutrient composition
of the diets before all trials was similar for all subjects.

Leucine Kinetics
Steady-state conditions were achieved during recovery from exercise as indicated
by plasma 13C-ketoisocaproate and breath 13CO2 enrichments (Figure 2). Leucine
Ra and NOLD were lower during recovery from milk-supplemented exercise than
with PLA (Ra 125.0 ± 20.3 vs. 154.2 ± 17.2 µmol∙kg–1∙h–1 and NOLD 89.4 ± 20.5
vs. 129.7 ± 17 µmol∙kg–1∙h–1, P < 0.05; Figure 3). Leucine Ox was higher after the
milk-supplemented run (35.6 ± 1.9 µmol∙kg–1∙h–1) than with either CHO (23.9 ±
3.7 µmol∙kg–1∙h–1) or PLA (24.5 ± 2.9 µmol∙kg–1∙h–1), P < 0.05. Leucine kinetics
did not differ between CHO and PLA.

Figure 2 — 13C-KIC plasma enrichments during 3 h recovery from a placebo- (PLA),

carbohydrate- (CHO), or milk- (MILK) supplemented endurance run.
462   Miller et al.

Figure 3 — 13CO2 breath enrichments during 3 h recovery from a placebo- (PLA),

carbohydrate- (CHO), or milk- (MILK) supplemented endurance run.

Discussion
Protein metabolism changes in response to submaximal endurance exercise (31,
38). During exercise there is an increase in protein breakdown and a decrease in
protein synthesis that are often countered by an increase in protein synthesis and
attenuated rates of breakdown during recovery (10). The purpose of this study was
to characterize how nutrient provision during exercise affects whole-body protein
utilization postexercise. Unlike previous supplementation trials, in which fairly
large portions of carbohydrate or nutrients were administered, a marginal caloric
load was used, taking into consideration the amount athletes would be willing to
ingest during training or a typical endurance event.
The major findings from this investigation were that consuming a carbohydrate-
protein supplement that provided 0.2 g∙kg–1∙h–1 CHO and 0.12 g∙kg–1∙h–1 protein
during exercise resulted in a decrease in leucine Ra (indicative of whole-body protein
breakdown), a decrease in NOLD (indicative of whole-body protein synthesis), and
an increase in Ox during the 3-h recovery period after a 120-min run at 65% VO2max.
Ingestion of carbohydrate alone (CHO) during exercise did not affect whole-body
leucine utilization during recovery.
Theoretically, maintaining blood glucose and glycogen stores in response to
carbohydrate ingestion should reduce the reliance on endogenous-protein break-
down to provide substrates for working muscle during prolonged exercise (3, 9,
14, 30). Previous studies have found a decrease in protein breakdown (30) and
attenuation in Ox (3, 9, 14) during exercise in response to carbohydrate intake.
Leucine oxidation during exercise was not measured in the current study, but
respiratory-exchange-ratio data collected during exercise in this study (21) showed
that drink composition did affect the fuel mix, with respiratory-exchange-ratio
values being higher during the CHO-supplemented run than during the PLA run.
 Leucine Kinetics During Recovery From Exercise   463

Despite differences in fuel utilization during exercise and higher plasma glucose
values postexercise (21), consumption of the CHO drink during exercise did
not differ from the PLA drink with regard to protein utilization during recovery
(Figure 4). It is important to note that the amount of carbohydrate we provided
(0.3 g∙kg–1∙h–1) was much less than that consumed in these previous reports (0.7
– 1.1 g∙kg–1∙h–1) (3, 14, 30). This might have contributed to the absence of an effect
of carbohydrate intake on protein utilization in the current study.
Few studies have evaluated the role of protein in sports drinks for endurance
athletes. Results from these studies suggest that glycogen storage and muscle
recovery are enhanced and that subsequent exercise performance is improved when
protein is added to recovery supplements (12, 13, 37, 41). Gains in whole-body and
leg-muscle protein are improved with protein consumption postexercise (17). The
addition of 10 g of protein to a carbohydrate-fat supplement postexercise increased
leg protein synthesis 6-fold and shifted whole-body protein net balance from nega-
tive to positive (17), which supports the notion that protein supplementation might
provide some benefit to endurance athletes.
Timing of protein ingestion is an important factor regarding its impact on
protein utilization (18, 28). Levenhagen et al. (18) found that consuming a protein-
carbohydrate mixture immediately after exercise improved net protein balance in the
recovery period. Similarly, Roy et al. observed improved nitrogen balance, weight
maintenance, and subsequent performance when a protein-containing supplement
was consumed immediately postexercise compared with the response noted when

Figure 4 — Whole-body protein turnover in trained men after a placebo- (PLA),

carbohydrate- (CHO), or milk- (MILK) supplemented endurance run. Leucine rate of appear-
ance (Ra), nonoxidative leucine disposal (NOLD), and oxidation (Ox) during recovery from
endurance exercise. Values are means ± standard error of the mean for 5 subjects. *Different
from PLA. †Different from CHO. P < 0.05.
464   Miller et al.

the supplement was consumed several hours before endurance exercise (28).
The observed decrease in proteolysis during recovery from a milk-supplemented
endurance-exercise session suggests that providing exogenous amino acids through-
out the run might have minimized dependence on endogenous-protein stores during
exercise. We previously reported (21) that branched-chain amino acid levels were
maintained during the milk- but not CHO- or PLA-supplemented runs. Protein
synthesis, as reflected by NOLD, was also attenuated during recovery after the
milk-supplemented exercise. The increase noted in Ox might partially explain the
latter observation. Because kinetic assessments were not made during the exercise
session, we postulate that dietary amino acids consumed during the run might have
been preferentially oxidized, thus decreasing their availability for whole-body
protein synthesis during the recovery period (35).
Koopman et al. (14) compared the effects on whole-body protein utilization
during recovery of consuming protein and carbohydrate or carbohydrate alone
during prolonged endurance exercise. Those authors used a higher level of pro-
tein than that of the current study and found that the addition of protein led to
decreased rates of protein breakdown during the recovery period without a decrease
in synthesis. Despite an increase in Ox in response to protein ingestion, protein
supplementation resulted in a more positive net leucine balance than did ingestion
of carbohydrate alone. In contrast, we observed suppression in both breakdown
and synthesis after protein intake compared with carbohydrate alone. Although
discrepancies in study findings are likely a result of differences in the quantity of
protein ingested, it is important to note that the exercise protocols also differed
between this study (2 h at 65% VO2max) and that of Koopman et al. (6 h at 50%
VO2max). Perhaps greater protein content in the milk supplement would have pro-
vided sufficient amino acids to not only support the increase in oxidation but also
enable an increase in protein synthesis. Potentially, the amount of protein needed
for this to occur would fall between the amount consumed in the present study
(~8 g/h) and that consumed by subjects in the study by Koopman et al. (~36 g/h)
(14). Practically speaking, determining the level of protein required to elicit this
desired response is very important to endurance athletes looking to optimize protein
utilization for the purpose of maintaining lean body mass throughout training and
the competitive season.
In summary, when compared with placebo, we observed a decrease in whole-
body protein breakdown and synthesis and an increase in Ox as evidenced by
leucine kinetics during the recovery period after a milk-supplemented endurance
run. Perhaps these data suggest a need for greater protein intake during exercise
to observe favorable results with respect to whole-body protein utilization, given
that the quantity provided in the current study was enough to elicit an increase in
oxidation and suppress breakdown but not enough to support synthesis. Carbohy-
drate ingestion did not influence whole-body protein turnover in the current study.
Conclusions from these observations are limited in that the 1-13C-leucine tracer
model provides an index of whole-body protein metabolism and is not reflective
of specific metabolic events occurring in muscle (27). Future studies are needed
to determine the role of protein supplementation during endurance exercise in
modulating skeletal-muscle protein turnover. Practically speaking, maintaining
lean body mass and strength throughout the course of a vigorous training program
is critical to endurance athletes’ performance. Nutritional supplementation might
 Leucine Kinetics During Recovery From Exercise   465

be a means by which this could be accomplished in this population. Benefits of

nutritional, particularly protein, supplementation must be convincingly demon-
strated to justify efforts to change an athlete’s training beliefs and behavior with
regard to this particular macronutrient. For competitive athletes seeking ways
to maintain lean body mass, nutritional supplementation during training might
prove to be a successful strategy in optimizing protein utilization after prolonged
endurance exercise.

Acknowledgments
The authors would like to extend their sincere gratitude to the following individuals who
assisted in execution of study protocols, sample collection, and sample processing:
Tabitha Elliott, Stavros Kavouras, Shannon Lennon, Timothy Scheett, Melissa Roti,
and John Youngdahl. This study was supported by the National Dairy Council and the
University of Connecticut Research Foundation.

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